Several lines of evidence suggest that erythropoietin, as a member of the cytokine superfamily, performs important physiologic functions which are mediated through interaction with the erythropoietin receptor (EPO-R). These actions include production of red blood cells, mitogenesis, modulation of calcium influx into smooth muscles and neural cells, and effects on intermediary metabolism.
EPO-R is a 66 kDa protein, and is part of the Type-1 cytokine receptor family. This family includes receptors for interleukin (IL)-IL2, IL3, IL4, IL5, IL6, IL7, IL9, IL11, granulocyte macrophage—colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Leukaemia Inhibiting Factor (LIF), Ciliary Neurotrophic Factor (CNTF), Thrombopoietin, Growth Hormone and Prolactin. These receptors are grouped together because of the homology of their extracellular domains. The conserved extracellular domain of these receptors has a length of approximately 200 amino acids, which contains four positionally conserved cysteine residues in the amino-terminal region (Cys 294, Cys 283, Cys 248, Cys 238) and a Trp-Ser-X-Trp-Ser motif located proximal to the transmembrane domain. The four cysteines appear to be critical to the maintenance and the structural integrity of the receptors (Murray, 1996, Harpers Biochemistry 24th ed. pp. 524-526, Appilion & Lange, Ltd.; Caravella et al., 1996, Protein: Struct. Funct. Gen. 24:394-401;).
Like many of the receptors within the Type-1 cytokine receptor family, the EPO-R appears to be activated when it is activated by its interaction with EPO. The first EPO-R in the dimer binds to EPO with a high affinity and the second EPO-R then binds to the complex with a low affinity. This dimerization of the EPO-R puts the Jak2 tyrosine kinases associated with EPO-R in close association, inducing their transphosphorylation. This activation leads to the tyrosine phosphorylation of several proteins that subsequently activate several different pathways, such as phosphatidylinositol (PI) 3-kinase pathway, the Ras/MAP kinase pathway, and the STAT pathway. These pathways trigger the physiological functions mediated by erythropoietin (Kirito et al., 2002, Blood 99:102-110; Livnah et al., 1999, Science 283:987-990; Naranda et al., 2002, Endocrinology 143:2293-2302; Remy et al., 1999, Science 283:990-993; and Yoshimura et al., 1996, The Oncol. 1:337-339).
In addition to forming multimers, many of the members of the type-1 family incorporate one of three different signal transducing receptor components—gp130, beta common (βc receptor), or the gamma subunit of the IL2 receptor (γc receptor)—in to their receptor complexes. For example, the receptor complex for GM-CSF consists of GM-CSF receptor, two βc receptors, and the GM-CSF ligand. The EPO-R has been known to form a complex with the βc receptor (See Yutaka et al., 1995, Bioch. Biophys. Res. Com. 208:1060-1066; Jubinsky et al., 1997, Blood 90:1867-1873; D'Andrea et al., 1998, J. Clin, Invest. 102:1951-1960). However, it has been reported that these complexes failed to result in any physiologically relevant effect (Scott et al., 2000, Blood, 96:1588-1590).
Recently a class of tissue protective cytokines, chemical or genetically modified erythropoietin molecules that demonstrate an enhanced tissue protective activity without any therapeutic erythropoietic effect have been disclosed. (See PCT Application No. PCT/US01/49479, U.S. patent application Ser. Nos. 10/188,905, 10/185,841, and 10/612,665, which are incorporated herein by reference herein in their entirety). These tissue protective cytokines protect, maintain, enhance and/or restore the function and/or viability of erythropoietin-responsive mammalian cells, tissues and organs, which include, but are not limited to, neuronal, retinal, muscle, heart, kidney cells or tissues. For example, these tissue protective cytokines have proven to be particularly effective in protecting against injury resulting from trauma and resulting inflammation to the brain (ischemic stroke, blunt trauma, subarrachnoid hemorrhage), spinal cord (ischemia, blunt force trauma), peripheral nerves (sciatic nerve injury, diabetic neuropathy, carpal tunnel syndrome), retinal (macular edema), and heart (myocardial infarct, chronic heart failure).
Unlike EPO, which binds the classical EPO-R dimer and provides a protective effect, it appears some modified EPO may not modulate the tissue protective activity by the same pathway since some of these tissue protective cytokines do not bind to the EPO-R homodimer. For example, a tissue protective cytokine generated by carbamylating erythropoietin in accordance with the procedure outlined in Example 2(B) of U.S. patent application Ser. No. 10/188,905 does not bind to the dimer EPO-R. (FIG. 1). The failure of the carbamylated tissue protective cytokine to bind to the EPO-R homodimer suggests that an alternative receptor or receptor complex mediates the tissue protective activity of these molecules.
It is towards this alternative receptor or receptor complex and the use of such alternative receptor or receptor complex as a means of screening potential compounds for tissue protective activities, that the present invention is directed.